Development and validation of an integrated cell culture-qRTPCR assay for simultaneous quantification of coxsackieviruses, echoviruses, and polioviruses in disinfection studies

摘要

This study demonstrated the applicability of integrated cell culture-quantitative RTPCRn(ICC-qRTPCR) for the simultaneous quantification of coxsackievirus, echovirus, and poliovirusnin disinfection studies. Buffalo green monkey cells were inoculated with a 10-fold dilution seriesnof mixed enteroviruses and incubated prior to qRTPCR quantification. Optimal assay conditionsnincluded three post infection washes and a 24-hour post infection incubation period based onnsuccessful differentiation between infectious and noninfectious viruses and significant andnconsistent viral replication rates. Ultraviolet disinfection studies were performed to validate thenICC-qRTPCR assay. Using the optimized assay, three-log microbial inactivation was achieved at UVndoses of 30–44, 28–42, and 28–29mJ/cm2nfor coxsackievirus B6, echovirus 12, and poliovirus 1,nrespectively. These results compare favorably to side-by-side assessments using conventionalncultural techniques and values previously reported in the literature. This indicates thatnICC-qRTPCR is a practical alternative for the simultaneous quantification of enterovirusesnin disinfection studies.